Robert McGill, 55, of Los Angeles is accused of killing his wife of five years, former high school sweetheart Shirley McGill, on July 14 during a Carnival Elation five-night cruise to Cabo San Lucas.
A federal defender entered the not guilty plea on McGill's behalf.
McGill spoke with his attorneys in hushed tones, in contrast to his initial court appearance on July 17 when he tried unsuccessfully to make a statement and was heard telling his attorneys that the victim's family needed to hear from him.
U.S. Magistrate Judge William McCurine Jr. scheduled another hearing for Monday.
McGill was indicted Wednesday, Assistant U.S. Attorney Joanna Curtis said.
She said she didn't seek a first-degree murder charge but refused to discuss her reasons, saying grand jury hearings are secret.
Curtis said she planned to talk to McGill's public defender to see if they can avoid a trial.
"I would like to resolve this case," Curtis said. "I know the family would like to resolve the case. It's up to the defendant and his lawyers."
The San Diego County medical examiner's office said Shirley McGill died of strangulation and blunt force injury to the head and torso.
McGill told authorities he "killed his wife in the bathroom of their cabin with his bare hands," court documents state.
McGill was a divorced father of two sons when he reconnected with Shirley through the Internet, colleagues said.
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